Bat coronaviruses related to SARS-CoV-2 and infectious for human cells.

The animal reservoir of SARS-CoV-2 is unknown despite reports of SARS-CoV-2-related viruses in Asian Rhinolophus bats1-4, including the closest virus from R. affinis, RaTG13 (refs. 5,6), and pangolins7-9. SARS-CoV-2 has a mosaic genome, to which different progenitors contribute. The spike sequence determines the binding affinity and accessibility of its receptor-binding domain to the cellular angiotensin-converting enzyme 2 (ACE2) receptor and is responsible for host range10-12. SARS-CoV-2 progenitor bat viruses genetically close to SARS-CoV-2 and able to enter human cells through a human ACE2 (hACE2) pathway have not yet been identified, although they would be key in understanding the origin of the epidemic. Here we show that such viruses circulate in cave bats living in the limestone karstic terrain in northern Laos, in the Indochinese peninsula. We found that the receptor-binding domains of these viruses differ from that of SARS-CoV-2 by only one or two residues at the interface with ACE2, bind more efficiently to the hACE2 protein than that of the SARS-CoV-2 strain isolated in Wuhan from early human cases, and mediate hACE2-dependent entry and replication in human cells, which is inhibited by antibodies that neutralize SARS-CoV-2. None of these bat viruses contains a furin cleavage site in the spike protein. Our findings therefore indicate that bat-borne SARS-CoV-2-like viruses that are potentially infectious for humans circulate in Rhinolophus spp. in the Indochinese peninsula.

SARS-CoV-2-related bat virus behavior in human-relevant models sheds light on the origin of COVID-19.

Bat sarbecovirus BANAL-236 is highly related to SARS-CoV-2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. BANAL-236 replicates efficiently and pauci-symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS-CoV-2. BANAL-236 infection leads to protection against superinfection by a virulent strain. We find no evidence of antibodies recognizing bat sarbecoviruses in populations in close contact with bats in which the virus was identified, indicating that such spillover infections, if they occur, are rare. Six passages in humanized mice or in human intestinal cells, mimicking putative early spillover events, select adaptive mutations without appearance of a furin cleavage site and no change in virulence. Therefore, acquisition of a furin site in the spike protein is likely a pre-spillover event that did not occur upon replication of a SARS-CoV-2-like bat virus in humans or other animals. Other hypotheses regarding the origin of the SARS-CoV-2 should therefore be evaluated, including the presence of sarbecoviruses carrying a spike with a furin cleavage site in bats.

Identification of Umbre Orthobunyavirus as a Novel Zoonotic Virus Responsible for Lethal Encephalitis in 2 French Patients with Hypogammaglobulinemia.

BACKGROUND: Human encephalitis represents a medical challenge from a diagnostic and therapeutic point of view. We investigated the cause of 2 fatal cases of encephalitis of unknown origin in immunocompromised patients. METHODS: Untargeted metatranscriptomics was applied on the brain tissue of 2 patients to search for pathogens (viruses, bacteria, fungi, or protozoans) without a prior hypothesis. RESULTS: Umbre arbovirus, an orthobunyavirus never previously identified in humans, was found in 2 patients. In situ hybridization and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) showed that Umbre virus infected neurons and replicated at high titers. The virus was not detected in cerebrospinal fluid by RT-qPCR. Viral sequences related to Koongol virus, another orthobunyavirus close to Umbre virus, were found in Culex pipiens mosquitoes captured in the south of France where the patients had spent some time before the onset of symptoms, demonstrating the presence of the same clade of arboviruses in Europe and their potential public health impact. A serological survey conducted in the same area did not identify individuals positive for Umbre virus. The absence of seropositivity in the population may not reflect the actual risk of disease transmission in immunocompromised individuals. CONCLUSIONS: Umbre arbovirus can cause encephalitis in immunocompromised humans and is present in Europe.

SARS-CoV-2 infection in schools in a northern French city: a retrospective serological cohort study in an area of high transmission, France, January to April 2020.

BackgroundChildren's role in SARS-CoV-2 epidemiology remains unclear. We investigated an initially unnoticed SARS-CoV-2 outbreak linked to schools in northern France, beginning as early as mid-January 2020.AimsThis retrospective observational study documents the extent of SARS-CoV-2 transmission, linked to an affected high school (n = 664 participants) and primary schools (n = 1,340 study participants), in the context of unsuspected SARS-CoV-2 circulation and limited control measures.MethodsBetween 30 March and 30 April 2020, all school staff, as well as pupils and their parents and relatives were invited for SARS-CoV-2 antibody testing and to complete a questionnaire covering symptom history since 13 January 2020.ResultsIn the high school, infection attack rates were 38.1% (91/239), 43.4% (23/53), and 59.3% (16/27), in pupils, teachers, and non-teaching staff respectively vs 10.1% (23/228) and 12.0% (14/117) in the pupils' parents and relatives (p < 0.001). Among the six primary schools, three children attending separate schools at the outbreak start, while symptomatic, might have introduced SARS-CoV-2 there, but symptomatic secondary cases related to them could not be definitely identified. In the primary schools overall, antibody prevalence in pupils sharing classes with symptomatic cases was higher than in pupils from other classes: 15/65 (23.1%) vs 30/445 (6.7%) (p < 0.001). Among 46 SARS-CoV-2 seropositive pupils < 12 years old, 20 were asymptomatic. Whether past HKU1 and OC43 seasonal coronavirus infection protected against SARS-CoV-2 infection in 6-11 year olds could not be inferred.ConclusionsViral circulation can occur in high and primary schools so keeping them open requires consideration of appropriate control measures and enhanced surveillance.

Prior infection by seasonal coronaviruses, as assessed by serology, does not prevent SARS-CoV-2 infection and disease in children, France, April to June 2020.

BackgroundChildren have a low rate of COVID-19 and secondary severe multisystem inflammatory syndrome (MIS) but present a high prevalence of symptomatic seasonal coronavirus infections.AimWe tested if prior infections by seasonal coronaviruses (HCoV) NL63, HKU1, 229E or OC43 as assessed by serology, provide cross-protective immunity against SARS-CoV-2 infection.MethodsWe set a cross-sectional observational multicentric study in pauci- or asymptomatic children hospitalised in Paris during the first wave for reasons other than COVID (hospitalised children (HOS), n = 739) plus children presenting with MIS (n = 36). SARS-CoV-2 antibodies directed against the nucleoprotein (N) and S1 and S2 domains of the spike (S) proteins were monitored by an in-house luciferase immunoprecipitation system assay. We randomly selected 69 SARS-CoV-2-seropositive patients (including 15 with MIS) and 115 matched SARS-CoV-2-seronegative patients (controls (CTL)). We measured antibodies against SARS-CoV-2 and HCoV as evidence for prior corresponding infections and assessed if SARS-CoV-2 prevalence of infection and levels of antibody responses were shaped by prior seasonal coronavirus infections.ResultsPrevalence of HCoV infections were similar in HOS, MIS and CTL groups. Antibody levels against HCoV were not significantly different in the three groups and were not related to the level of SARS-CoV-2 antibodies in the HOS and MIS groups. SARS-CoV-2 antibody profiles were different between HOS and MIS children.ConclusionPrior infection by seasonal coronaviruses, as assessed by serology, does not interfere with SARS-CoV-2 infection and related MIS in children.

Bio++: efficient extensible libraries and tools for computational molecular evolution.

Efficient algorithms and programs for the analysis of the ever-growing amount of biological sequence data are strongly needed in the genomics era. The pace at which new data and methodologies are generated calls for the use of pre-existing, optimized-yet extensible-code, typically distributed as libraries or packages. This motivated the Bio++ project, aiming at developing a set of C++ libraries for sequence analysis, phylogenetics, population genetics, and molecular evolution. The main attractiveness of Bio++ is the extensibility and reusability of its components through its object-oriented design, without compromising the computer-efficiency of the underlying methods. We present here the second major release of the libraries, which provides an extended set of classes and methods. These extensions notably provide built-in access to sequence databases and new data structures for handling and manipulating sequences from the omics era, such as multiple genome alignments and sequencing reads libraries. More complex models of sequence evolution, such as mixture models and generic n-tuples alphabets, are also included.

Performance of clinical metagenomics in France: a prospective observational study.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) allows untargeted identification of a broad range of pathogens, including rare or novel microorganisms. Despite the recognition of mNGS as a valuable diagnostic tool for infections, the most relevant indications for this innovative strategy remain poorly defined. We aimed to assess the determinants of positivity and clinical utility of mNGS. METHODS: In this observational study, we prospectively performed short-read shotgun metagenomics analysis as a second-line test (in cases of negative first-line test or when the symptoms were not fully explained by initial positive results) or as a first-line test in life-threatening situations requiring urgent non-targeted pathogen identification at the Necker-Enfants Malades Hospital (Paris, France). All sample types, clinical indications, and patient populations were included. Samples were accompanied by a mandatory form completed by the senior clinician or pathologist, on which the clinical level of suspected infection (defined as high or low) was indicated. We assessed the variables (gender, age, immune status, initial suspicion of infection, indication, and sample type) associated with mNGS pathogen detection using odds ratios (ORs) from multivariate logistic regression. Additional investigations were carried out using specific PCR or culture techniques, to confirm positive mNGS results, or when infectious suspicion was particularly high despite a negative mNGS result. FINDINGS: Between Oct 29, 2019, and Nov 7, 2022, we analysed 742 samples collected from 523 patients. The initial suspicion of infection was either high (n=470, 63%) or low (n=272, 37%). Causative or possibly causative pathogens were detected in 117 (25%) samples from patients with high initial suspicion of infection, versus nine (3%) samples analysed to rule out infection (OR 9·1, 95% CI 4·6-20·4; p<0·0001). We showed that mNGS had higher odds of detecting a causative or possibly causative pathogenic virus on CNS biopsies than CSF samples (4·1, 1·7-10·7; p=0·0025) and in samples from immunodeficient compared with immunocompetent individuals (2·4, 1·4-4·1; p=0·0013). Concordance with conventional confirmatory tests results was 103 (97%) of 106, when mNGS detected causative or possibly causative pathogens. Altogether, among 231 samples investigated by both mNGS and subsequent specific tests, discordant results were found in 69 (30%) samples, of which 58 (84%) were mNGS positive and specific tests negative, and 11 (16%) mNGS negative and specific tests positive. INTERPRETATION: Major determinants of pathogen detection by mNGS are immune status and initial level of suspicion of infection. These findings will contribute, along with future studies, to refining the positioning of mNGS in diagnostic and treatment decision-making algorithms. FUNDING: Necker-Enfants Malades Hospital and Institut Pasteur. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

TPMS: a set of utilities for querying collections of gene trees.

BACKGROUND: The information in large collections of phylogenetic trees is useful for many comparative genomic studies. Therefore, there is a need for flexible tools that allow exploration of such collections in order to retrieve relevant data as quickly as possible. RESULTS: In this paper, we present TPMS (Tree Pattern-Matching Suite), a set of programs for handling and retrieving gene trees according to different criteria. The programs from the suite include utilities for tree collection building, specific tree-pattern search strategies and tree rooting. Use of TPMS is illustrated through three examples: systematic search for incongruencies in a large tree collection, a short study on the Coelomata/Ecdysozoa controversy and an evaluation of the level of support for a recently published Mammal phylogeny. CONCLUSION: TPMS is a powerful suite allowing to quickly retrieve sets of trees matching complex patterns in large collection or to root trees using more rigorous approaches than the classical midpoint method. As it is made of a set of command-line programs, it can be easily integrated in any sequence analysis pipeline for an automated use.

Virus Pop-Expanding Viral Databases by Protein Sequence Simulation.

The improvement of our knowledge of the virosphere, which includes unknown viruses, is a key area in virology. Metagenomics tools, which perform taxonomic assignation from high throughput sequencing datasets, are generally evaluated with datasets derived from biological samples or in silico spiked samples containing known viral sequences present in public databases, resulting in the inability to evaluate the capacity of these tools to detect novel or distant viruses. Simulating realistic evolutionary directions is therefore key to benchmark and improve these tools. Additionally, expanding current databases with realistic simulated sequences can improve the capacity of alignment-based searching strategies for finding distant viruses, which could lead to a better characterization of the "dark matter" of metagenomics data. Here, we present Virus Pop, a novel pipeline for simulating realistic protein sequences and adding new branches to a protein phylogenetic tree. The tool generates simulated sequences with substitution rate variations that are dependent on protein domains and inferred from the input dataset, allowing for a realistic representation of protein evolution. The pipeline also infers ancestral sequences corresponding to multiple internal nodes of the input data phylogenetic tree, enabling new sequences to be inserted at various points of interest in the group studied. We demonstrated that Virus Pop produces simulated sequences that closely match the structural and functional characteristics of real protein sequences, taking as an example the spike protein of sarbecoviruses. Virus Pop also succeeded at creating sequences that resemble real sequences not included in the databases, which facilitated the identification of a novel pathogenic human circovirus not included in the input database. In conclusion, Virus Pop is helpful for challenging taxonomic assignation tools and could help improve databases to better detect distant viruses.

A Search for Tick-Associated, Bronnoya-like Virus Spillover into Sheep.

Tick-borne diseases are responsible for many vector-borne diseases within Europe. Recently, novel viruses belonging to a new viral family of the order Bunyavirales were discovered in numerous tick species. In this study, we used metatranscriptomics to detect the virome, including novel viruses, associated with Ixodes ricinus collected from Romania and France. A bunyavirus-like virus related to the Bronnoya virus was identified for the first time in these regions. It presents a high level of amino-acid conservation with Bronnoya-related viruses identified in I. ricinus ticks from Norway and Croatia and with the Ixodes scapularis bunyavirus isolated from a tick cell line in Japan in 2014. Phylogenetic analyses revealed that the Bronnoya viruses' sub-clade is distinct from several Bunyavirales families, suggesting that it could constitute a novel family within the order. To determine if Bronnoya viruses could constitute novel tick-borne arboviruses, a Luciferase immunoprecipitation assay for detecting antibodies in the viral glycoprotein of the Romanian Bronnoya virus was used to screen sera from small ruminants exposed to tick bites. No positive serum was detected, suggesting that this virus is probably not able to infect small ruminants. This study represents the first serological investigation of mammalian infections with a Bronnoya-like virus and an initial step in the identification of potential new emergences of tick-borne arboviruses.

Comprehensive Characterization of Viral Diversity of Female Mosquitoes in Madagascar.

The diversity and circulation of arboviruses are not much studied in Madagascar. The fact is that arboviral emergences are rarely detected. The existing surveillance system primarily relies on serological detection and records only a few human infections annually. The city of Mahajanga, however, experienced a confirmed dengue fever epidemic in 2020 and 2021. This study aimed to characterize and analyze the virome of mosquitoes collected in Mahajanga, near patients with dengue-like syndromes to detect known and unknown viruses as well as investigate the factors contributing to the relative low circulation of arboviruses in the area. A total of 4280 mosquitoes representing at least 12 species from the Aedes, Anopheles, and Culex genera were collected during the dry and the rainy seasons from three sites, following an urbanization gradient. The virome analysis of 2192 female mosquitoes identified a diverse range of viral families and genera and revealed different patterns that are signatures of the influence of the mosquito genus or the season of collection on the composition and abundance of the virome. Despite the absence of known human or veterinary arboviruses, the identification and characterization of viral families, genera, and species in the mosquito virome contribute to our understanding of viral ecology and diversity within mosquito populations in Madagascar. This study serves as a foundation for ongoing surveillance efforts and provides a basis for the development of preventive strategies against various mosquito-borne viral diseases, including known arboviruses.

Genotype and Phenotype Characterization of Rhinolophus sp. Sarbecoviruses from Vietnam: Implications for Coronavirus Emergence.

Bats are a major reservoir of zoonotic viruses, including coronaviruses. Since the emergence of SARS-CoV in 2002/2003 in Asia, important efforts have been made to describe the diversity of Coronaviridae circulating in bats worldwide, leading to the discovery of the precursors of epidemic and pandemic sarbecoviruses in horseshoe bats. We investigated the viral communities infecting horseshoe bats living in Northern Vietnam, and report here the first identification of sarbecoviruses in Rhinolophus thomasi and Rhinolophus siamensis bats. Phylogenetic characterization of seven strains of Vietnamese sarbecoviruses identified at least three clusters of viruses. Recombination and cross-species transmission between bats seemed to constitute major drivers of virus evolution. Vietnamese sarbecoviruses were mainly enteric, therefore constituting a risk of spillover for guano collectors or people visiting caves. To evaluate the zoonotic potential of these viruses, we analyzed in silico and in vitro the ability of their RBDs to bind to mammalian ACE2s and concluded that these viruses are likely restricted to their bat hosts. The workflow applied here to characterize the spillover potential of novel sarbecoviruses is of major interest for each time a new virus is discovered, in order to concentrate surveillance efforts on high-risk interfaces.

Microseek: A Protein-Based Metagenomic Pipeline for Virus Diagnostic and Discovery.

We present Microseek, a pipeline for virus identification and discovery based on RVDB-prot, a comprehensive, curated and regularly updated database of viral proteins. Microseek analyzes metagenomic Next Generation Sequencing (mNGS) raw data by performing quality steps, de novo assembly, and by scoring the Lowest Common Ancestor (LCA) from translated reads and contigs. Microseek runs on a local computer. The outcome of the pipeline is displayed through a user-friendly and dynamic graphical interface. Based on two representative mNGS datasets derived from human tissue and plasma specimens, we illustrate how Microseek works, and we report its performances. In silico spikes of known viral sequences, but also spikes of fake Neopneumovirus viral sequences generated with variable evolutionary distances from known members of the Pneumoviridae family, were used. Results were compared to Chan Zuckerberg ID (CZ ID), a reference cloud-based mNGS pipeline. We show that Microseek reliably identifies known viral sequences and performs well for the detection of distant pseudoviral sequences, especially in complex samples such as in human plasma, while minimizing non-relevant hits.

Detection of Phenuiviridae, Chuviridae Members, and a Novel Quaranjavirus in Hard Ticks From Danube Delta.

Ticks are involved in the transmission of various pathogens and several tick-borne diseases cause significant problems for the health of humans and livestock. The members of the Quaranjavirus genus are mainly associated with argas ticks but recent studies demonstrated the presence of novel quaranjaviruses-like in ixodid ticks. In 2020, 169 Rhipicephalus sanguineus ticks were collected in Southern Romania from small ruminants and analyzed by high-throughput transcriptome sequencing. Among the viral families that infect Romanian ticks, we have identified sequences from Phenuiviridae (Brown dog tick phlebovirus 1 [BDTPV1] and Brown dog tick phlebovirus 2 [BDTPV2]) and Chuviridae families (Cataloi mivirus [CTMV]), and numerous sequences from a new quaranjavirus-like, tentatively named Cataloi tick quaranjavirus (CTQV). Phylogenetic analyses performed on the five segments show that CTQV is phylogenetically positioned within a clade that encompasses Ixodidae-borne viruses associated with iguanas, small ruminants, seabirds, and penguins distributed across different geographical areas. Furthermore, CTQV is positioned differently depending on the segment considered. This is the first report on the detection of a quaranjavirus-like in Eastern Europe. Further investigations are needed to discern its infectivity and pathogenicity against vertebrates.

The virome of Rhipicephalus, Dermacentor and Haemaphysalis ticks from Eastern Romania includes novel viruses with potential relevance for public health.

Ticks are involved in the transmission of various pathogens and several tick-borne diseases cause significant problems for the health of humans and livestock. The composition of viral communities in ticks and their interactions with pathogens, is poorly understood, particularly in Eastern Europe, an area that represents a major hub for animal-arthropod vectors exchanges (e.g., via bird migrations). The aim of this study was to describe the virome of Dermacentor sp., Rhipicephalus sp. and Haemaphysalis sp. ticks collected from relatively little studied regions of Romania (Iasi and Tulcea counties) located at the intersection of various biotopes, countries and routes of migrations. We also focused the study on viruses that could potentially have relevance for human and animal health. In 2019, more than 500 ticks were collected from the vegetation and from small ruminants and analysed by high-throughput transcriptome sequencing. Among the viral communities infecting Romanian ticks, viruses belonging to the Flaviviridae, Phenuiviridae and Nairoviridae families were identified and full genomes were derived. Phylogenetic analyses placed them in clades where mammalian isolates are found, suggesting that these viruses could constitute novel arboviruses. The characterization of these communities increase the knowledge of the diversity of viruses in Eastern Europe and provides a basis for further studies about the interrelationship between ticks and tick-borne viruses.

Deep Impact of Random Amplification and Library Construction Methods on Viral Metagenomics Results.

Clinical metagenomics is a broad-range agnostic detection method of pathogens, including novel microorganisms. A major limit is the low pathogen load compared to the high background of host nucleic acids. To overcome this issue, several solutions exist, such as applying a very high depth of sequencing, or performing a relative enrichment of viral genomes associated with capsids. At the end, the quantity of total nucleic acids is often below the concentrations recommended by the manufacturers of library kits, which necessitates to random amplify nucleic acids. Using a pool of 26 viruses representative of viral diversity, we observed a deep impact of the nature of sample (total nucleic acids versus RNA only), the reverse transcription, the random amplification and library construction method on virus recovery. We further optimized the two most promising methods and assessed their performance with fully characterized reference virus stocks. Good genome coverage and limit of detection lower than 100 or 1000 genome copies per mL of plasma, depending on the genome viral type, were obtained from a three million reads dataset. Our study reveals that optimized random amplification is a technique of choice when insufficient amounts of nucleic acid are available for direct libraries constructions.

Viromics on Honey-Baited FTA Cards as a New Tool for the Detection of Circulating Viruses in Mosquitoes.

Worldwide, emerging and re-emerging infectious diseases (EIDs) are a major burden on public and animal health. Arthropod vectors, with mosquitoes being the main contributors of global disease, transmit more than 70% of the recognized EIDs. To assess new alternatives for arthropod-borne viral diseases surveillance, and for the detection of new viruses, honey-baited Flinders Technology Associates (FTA) cards were used as sugar bait in mosquito traps during entomological surveys at the Llobregat River Delta (Catalonia, Spain). Next generation sequencing (NGS) metagenomics analysis was applied on honey-baited FTA cards, which had been exposed to field-captured mosquitoes to characterize their associated virome. Arthropod- and plant-infecting viruses governed the virome profile on FTA cards. Twelve near-complete viral genomes were successfully obtained, suggesting good quality preservation of viral RNAs. Mosquito pools linked to the FTA cards were screened for the detection of mosquito-associated viruses by specific RT-PCRs to confirm the presence of these viruses. The circulation of viruses related to Alphamesonivirus, Quaranjavirus and unclassified Bunyavirales was detected in mosquitoes, and phylogenetic analyses revealed their similarities to viruses previously reported in other continents. To the best our knowledge, our findings constitute the first distribution record of these viruses in European mosquitoes and the first hint of insect-specific viruses in mosquitoes' saliva in field conditions, demonstrating the feasibility of this approach to monitor the transmissible fraction of the mosquitoes' virome. In conclusion, this pilot viromics study on honey-baited FTA cards was shown to be a valid approach for the detection of viruses circulating in mosquitoes, thereby setting up an alternative tool for arbovirus surveillance and control programs.

RNA Viruses of Amblyomma variegatum and Rhipicephalus microplus and Cattle Susceptibility in the French Antilles.

Ticks transmit a wide variety of pathogens including bacteria, parasites and viruses. Over the last decade, numerous novel viruses have been described in arthropods, including ticks, and their characterization has provided new insights into RNA virus diversity and evolution. However, little is known about their ability to infect vertebrates. As very few studies have described the diversity of viruses present in ticks from the Caribbean, we implemented an RNA-sequencing approach on Amblyomma variegatum and Rhipicephalus microplus ticks collected from cattle in Guadeloupe and Martinique. Among the viral communities infecting Caribbean ticks, we selected four viruses belonging to the Chuviridae, Phenuiviridae and Flaviviridae families for further characterization and designing antibody screening tests. While viral prevalence in individual tick samples revealed high infection rates, suggesting a high level of exposure of Caribbean cattle to these viruses, no seropositive animals were detected. These results suggest that the Chuviridae- and Phenuiviridae-related viruses identified in the present study are more likely tick endosymbionts, raising the question of the epidemiological significance of their occurrence in ticks, especially regarding their possible impact on tick biology and vector capacity. The characterization of these viruses might open the door to new ways of preventing and controlling tick-borne diseases.

Whole genome sequencing and phylogenetic characterization of a novel bat-associated picornavirus-like virus with an unusual genome organization.

The order Picornavirales is one of the most important viral orders in terms of virus diversity and genome organizations, ranging from a mono- or bi-cistronic expression strategies to the recently described poly-cistronic Polycipiviridae viruses. We report here the description and characterization of a novel picorna-like virus identified in rectal swabs of frugivorous bats in Cambodia that presents an unusual genome organization. Kandabadicivirus presents a unique genome architecture and distant phylogenetic relationship to the proposed Badiciviridae family. These findings highlight a high mosaicism of genome organizations among the Picornavirales.